The use of multiphoton laser scanning microscopy (MPLSM) to improve intravital imaging in physiologically relevant animal models holds particular promise for cancer studies. MPLSM has many advantages for intravital imaging, including high resolution, deep sectioning, and improved tissue viability. Moreover, MPLSM when coupled with second harmonic, fluorescent lifetime and spectral imaging approaches holds the promise of exploiting intrinsic sources of contrast that obviate the need for exogenous labels. Currently, there are no commercial MPLSM systems on the market that are well suited for this task in vivo. The overall objective of the research aims is to add new multimodality functionality to the Prairie Technologies Ultima IV MPSLM for intravital microscopy in animal models of carcinoma progression. This instrument will permit imaging of tumor cells invading into local stroma, analysis of changes in the collagen stroma by second harmonic generation (SHG), visualization of metabolic changes that accompany tumor growth and progression, and investigation of signaling molecules in vivo, especially those that may be relevant to tumor growth, survival, progression, invasion, or metastasis. We propose the following specific Aims, through which we will augment the capabilities of the Ultima to achieve these goals. 1. Develop optical and software approaches for SHG signal collection and analysis 2. Develop an new acquisition system for the Ultima IV that will consist of new software and hardware for simultaneous intensity, spectral and lifetime visualization. 3. Develop an adaptive optics system for correct system aberrations to enable image acquisition from deeper within living specimens. These animal studies will pave the technology for future development of imaging and visualization approaches that have the potential to improve the diagnosis and staging of human disease. We envision that the development of a user-friendly, turn-key multiphoton microscope will facilitate the use of this technology by pathologists. Moreover, these technologies could be a future adjunct to surgery. The understanding of tumor progression that will result from imaging animal models will have a positive impact on targeting future therapies. PUBLIC HEALTH RELEVANCE: The development of improved methods for in vivo multiphoton imaging will help identify key signatures of malignancy of cancer. Understanding the underlying mechanisms of cancer progression in animal models can suggest future targets for human therapy.